Cholesterol and glycolipids are vital lipid molecules for the maintenance of cellular membranes. They are supplied to the cell by de novo synthesis and exogenous endocytic uptake of lipoproteins. The continuous recycling of these lipid molecules and maintenance of lipid equilibrium is crucial for cell viability. Receptors, enzymes and lipid transport molecules all play a role in maintenance of cellular membrane-lipid concentrations. In many inherited, human diseases there is an imbalance in trafficking of lipoprotein or plasma membrane-derived lipid molecules and they accumulate in cellular endocytic compartments resulting in a variety of lipidoses commonly called lysosomal storage diseases. Our investigations of the cellular pathology in Niemann Pick C disease, a cholesterol storage disease, has revealed that the late endocytic pathway accumulates excess cholesterol due to lack of, or mutations in Niemann Pick 1 or Niemann Pick 2 proteins. Studies on the expression and trafficking of functional NPC1-GFP in living normal fibroblasts revealed that the late endocytic pathway is composed of rapidly moving tubules interacting with lysosomes. NPC1, a membrane spanning protein with a sterol sensing domain that binds cholesterol, resides in membranes of the late endosomal tubular network and thus can be used to visualize the activity this endocytic compartment in living cells. NPC2 a smaller, soluble protein, traffics in late endosomal tubules to lysosomes where it resides in the lumen of this compartment. Studies on the expression of a non-functional mutant NPC1-GFP in living Niemann-Pick C fibroblasts showed that the late endosomal tubules are immobile and thus not functioning in trafficking of lipid molecules. We then showed a correlation between the mobility of late endosomal tubules and cholesterol clearance from lysosomes in mutant cells by expressing WT NPC1 in mutant cells or by depleting cholesterol nutritionally from mutant cells. These studies led to the hypothesis that cells from other lysosomal lipid storage diseases such as Tay Sachs, Fabry and Gaucher might have similar defective late endosomal tubular mobility due to pathological accumulation of lipids in these inherited diseases. Using expression of NPC1-GFP or uptake of fluorescent dextran to mark the late endosomal tubular compartment, immobility of late endosomal tubules was found to be a pathology common to these inherited glycolipid storage diseases. As with Niemann Pick C cells, expression of NPC1 or nutritional depletion of cholesterol restored mobility of late endosomal tubules resulting in egress of accumulated glycolipids from lysosomes in the various mutant cells. We are investigating the cellular fate of the glycolipids released from lysosomes by the induction of mobility of late endosomal tubules in mutant cells (Tays Sachs? Fabrey and Gaucher) that have inadequate glycolipid hydrolyzing enzyme activity. Our studies show that maintenance of late endosomal tubular mobility is critical for the normal egress of a variety of lipid molecules from lysosomes. Exogeneously derived lipids trafficking to lysosomes for turnover may accumulate in these organelles as a result of interruption of the rapidly occurring fusion and/or fission events that occur to effect molecular exchange between late endosomal tubules and lysosomes. Our fluorescence studies in living cells suggest the existence of a hybrid compartment containing both late endosomal and lysosomal components in mutant cells but not in normal cells, consistent with the concept of delayed and faulty interaction between these organelles. We question what molecular deficiencies or excesses occur in late endocytic membranes to interrupt the fusion/ fission events. Studies are currently in progress to characterize the abnormal hybrid compartment found in mutant cells. A cholesterol-loaded endocytic compartment (CLC) free of Golgi, endoplasmmic reticulum and plasma membrane has been isolated by density gradient centrifugation, from normal and mutant human diploid fibroblasts fed high levels of low density lipoprotein (LDL). This CLC fraction contains mostly elongated membranous tubules as visualized with electron microscopy. Protein analysis of the CLC fractions reveals that these tubules from the NPC1 null mutant cells lack AP-3, the adaptor complex required for endocytic trafficking of lysosomal membrane proteins (LAMP-1 and -2 and LIMP-1). Further analysis may reveal the scarcity of other proteins in the CLC fraction causing, or resulting from, the formation of a hybrid late endocytic organelle. Knowledge of lipids and proteins critical to preventing the pathological stasis of the late endosomal tubular/lysosomal interaction will have an impact on therapies designed to correct inherited metabolic diseases that disrupt cellular membrane lipid equilibrium.